I am an MD/PhD student with the long-term goal of becoming a physician-scientist in the field of pediatric hematology. This application describes my PhD thesis plans to investigate the mechanisms of red blood cell development. The maturation of bone marrow precursors into mature red blood cells requires dramatic morphological and physiological changes directed toward the ultimate function of oxygen transport. One interesting and poorly understood aspect of mammalian erythropoiesis is a series of specialized cell divisions that culminate in expulsion of the nucleus to generate mature anucleate erythrocytes, believed to improve the rheology of blood flow. The goal of my research is to elucidate the molecular and cellular mechanisms governed by Trim58, a putative E3 ubiquitin ligase that facilitates erythroblast enucleation. Trim58 is an erythroid protein induced o high levels during terminal maturation. Genome wide association studies (GWAS) implicate a role for the human TRIM58 gene in erythropoiesis. Our preliminary studies show that Trim58 knockdown inhibits nuclear extrusion and enucleation of murine fetal erythroblasts. Furthermore, Trim58 interacts with the molecular motor dynein to induce its proteasome-dependent degradation. Dynein and kinesin are directionally opposed motors that transport cargo, including nucleii, along microtubules. I hypothesize that Trim58 promotes erythroblast enucleation by degrading dynein to allow kinesin-mediated polarization of the nucleus to one side of the cell, thereby facilitating its expulsion. I will investigate the role of Trim58 during terminal erythroid maturation and define its activity as an E3 ubiquitin ligase. In Aim 1, I will ue live-cell imaging and imaging-flow cytometry to test whether Trim58 and kinesins are necessary for nuclear polarization. I will generate shRNAs targeting dynein heavy chain and kinesin heavy chain (Kif5b) and investigate the effects of molecular motor imbalance on erythroid maturation in the absence or presence Trim58. In Aim 2, I will use both in vitro (with purified recombinant protein) and cell culture systems to test the E3 ligase activity of Trim58 to ubiquitylate itself ad dynein. I will also identify novel substrates of Trim58 using an affinity purification-mass spectrometry technique. Studying the actions of Trim58 during erythroid maturation, including its E3 ubiquitin ligase activity and potential substrates, will provide insight into mechanisms of normal erythropoiesis. My studies are relevant to the pathophysiology of numerous anemias associated with defective terminal maturation and may enhance current efforts to generate mature red blood cells ex vivo for transfusion therapies.